Introduction L-Type voltage-operated Ca2+ channels provide the route for bulk Ca2+ influx to promote rhythmic uterine contractions during labour. However, they are not the only mediator of Ca2+ entry into myometrial cells. TRPV4, a non-selective cation channel, is hypothesized to be capable of activating spontaneously in myometrial cells in the absence of agonist stimulation. This study investigated the contribution of TRPV4 channels to spontaneous and oxytocin stimulated contractions in human pregnant myometrium in vitro. Methods Twenty-two biopsies of human myometrium were obtained, with written informed consent, from term (38.6 ±1.6 weeks) non-labouring pregnant women during elective caesarean section (Ethics No. EC00/137). Tissues were dissected and mounted in an organ bath, immersed in physiological salt solution and placed under 29.34mN of tension to establish spontaneous contractions. Subsequently, myometrial strips were exposed to cumulative concentrations (1nM-10µM) of TRPV4 agonist (GSK101 – GSK1016790A) or antagonist (RN17 – RN1734), at 60 min intervals. Myometrial tissues were also pre-treated (60 min) with RN17 (200nM or 1µM) and subsequently treated with GSK101 or oxytocin (1nM-10µM). Data are given as mean ± S.E.M, a p value ≤ 0.05 was deemed statistically significant. Data were analysed using two-way ANOVA with Bonferroni correction to determine differences between treatment and DMSO control groups (% response MIT – mean integral tension or amplitude). Results GSK101 produced a dose-dependent inhibitory effect on amplitude (2.30% ±0.45, n=7) and MIT (8.01% ±2.55, n=7) of spontaneous contraction of human myometrium vs control group (n=7). GSK101 (TRPV4 agonist) had the opposite effect to that expected; it decreased contraction amplitude by 33.61% ±7.35 (n=7, p=0.0006) and MIT by 27.53% ±10.17 (n=7, p=0.0191) vs control group (n=7). Pre-treatment of myometrial tissues with RN17 reduced subsequent oxytocin stimulated contractions [% MIT by 47.16 ±27.10, (200nM RN17 +oxytocin, n=4) and 74.32 ±22.14 (1µM, n=4) vs control group (n=3)]. The % change of the amplitude of tissues pre-treated with 200nM and 1µM of RN17 was 19.60 ±9.77 (n=4) and 6.18 ±20.46 (n=4), respectively. Pre-incubation of myometrial strips with 1µM RN17 reduced the inhibitory effect of GSK101 on the % MIT by 42.02 ±15.68 (n=4), reaching statistical significance at 10µM of GSK101 (p=0.0071). Pre-incubation with 200nM RN17 also reduced the inhibitory effect of GSK101 by 71.25% ±11.70, reaching statistical significance at 1µM (p= 0.0010) and 10µM (p= 0,0084). Conclusions Using the antagonist RN17, these results demonstrate TRPV4 modulates spontaneous and oxytocin-induced contractility in human myometrium. The TRPV4 agonist GSK101 supressed myometrium contractions; potentially through activation of BK channels or off-target effects. Funding: NIH R01 HD092316; Tommy’s Charity